Project Summary As ubiquitous and highly conserved molecular chaperones, Hsp70s play multiple essential roles in maintaining cellular protein homeostasis (proteostasis) through assisting in protein folding, assembly, degradation, disaggregation, and transportation across membrane. The fundamental importance of maintaining proteostasis inevitably links Hsp70s with many life-threatening human diseases, most notably cancers and neurodegenerative disorders. Thus, elucidating the biochemical and structural properties of Hsp70s will not only advance our understanding on the basic molecular mechanism of Hsp70-assited folding, but also provide a crucial and solid foundation for rational design of novel therapeutics for cancers and neurodegenerative disorders. All Hsp70s contain two functional domains, a nucleotide binding domain (NBD) and a substrate- binding domain (SBD), corresponding to two key intrinsic biochemical activities: ATPase and polypeptide substrate binding. Although NBD and SBD can each bind their substrates independently, the chaperone activity of Hsp70s strictly requires the tight coupling of these two domains upon ATP binding. The current paradigm for Hsp70 chaperone cycle was proposed primarily based on this essential allosteric coupling, which is mainly about how different nucleotide-bound states control polypeptide substrate binding. In spite of extensive efforts, the very basic mechanisms of Hsp70-assisted protein folding are still ill-defined due to a lack of in-depth understanding of two key questions: 1) Both binding and release of polypeptide substrates were proposed to occur in the ATP-bound state (Hsp70-ATP). How does Hsp70-ATP decide when to bind and when to release substrates to promote a productive chaperone cycle? and 2) Until now, no structure is available for an Hsp70-ATP with a polypeptide substrate bound. How does Hsp70-ATP bind polypeptide substrates to promote productive protein folding? Thus, the overall objective of this proposal is to analyze these two key questions in order to dissect the basic molecular mechanisms of Hsp70 chaperone function. Recently, we have successfully solved three Hsp70-ATP structures and unexpectedly revealed two novel and completely different conformations of the polypeptide-binding pocket, suggesting that the polypeptide-binding pocket of the ATP- bound state is highly dynamic. More importantly, our solution studies inspired by these structures have revolutionized the well-established chaperone cycle with three paradigm-shifting discoveries: 1) an active release of bound substrate upon ATP-binding, 2) Hsp40 co-chaperone is the key to initiate efficient substrate binding to Hsp70-ATP and thus start productive chaperone cycle, and 3) an active unfolding by Hsp70?s polypeptide-binding pocket. Based on these original discoveries, we propose the following two Specific Aims: 1) Characterize the dynamics of Hsp70s? polypeptide-binding pocket in the active chaperone cycle, and 2) Determine the molecular mechanism of polypeptide substrate binding to Hsp70-ATP. To achieve our goal, we use a multidisciplinary approach combining biochemistry, X-ray crystallography, FRET, single-molecule biophysics, EPR, computational chemistry, and yeast and E.coli genetics. We expect that successful completion of this proposal will help us realize our long-term goal, which is to establish a thorough mechanism understanding of the very basic mechanism of Hsp70 chaperone activity in maintaining proteostasis.